We have continued our studies of chromatin structure in the neighborhood of expressed genes. The globin gene family in chicken erythroid cells serves as a model system in which it is possible to study the mechanisms associated with regulation of the cluster and individual members of the family during erythroid development. We have focused attention on the 1.2 kb insulator DNA sequence at the 5' end of the chicken beta-globin locus. This insulator is capable both of blocking the influence of outside enhancers and of preventing the encroachment of condensed chromatin that might shut down expression of the entire region. We had earlier narrowed down enhancer blocking activity to a small DNA fragment within the 1.2 kb sequence, and shown that this activity involves binding of the ubiquitous vertebrate nuclear protein CTCF. We showed that the CTCF binding site also plays an important role in imprinting at the Igf2/H19 locus in mouse and humans. Now we have found that similar sites flank not only the chicken beta-globin locus, but also the beta-globin loci of mouse and human. In the human locus the downstream site is in a position to block endogenous enhancers that might otherwise disrupt the program of globin gene expression. We have been exploring the mouse and human loci to search for additonal regulatory elements. The insulator also has the ability to protect against position effects reporter genes that are stably transfected into cell lines or animals. We have now shown that this protective ability is present in a 'core' element, 250 bp long, from within the 1.2 kb insulator. Deletion of subregions within the core shows that the CTCF site is not involved in this activity, but four other subregions corresponding to binding sites for as yet unidentified nuclear proteins are important for position effect protection. These results mean that it is now possible to separate two aspects of insulator activity - enhancer blocking and position effect protection-and study them in isolation. We have also been studying the pattern of chromatin structural changes and modifications over the globin locus and its neighborhood during erythroid development. We had found earlier that an erythroid specific folate receptor (FR) gene lies upstream of the chicken beta-globin locus, separated from it by 16 kilobases of condensed non-expressed chromatin. Making use of high resolution immunoprecipitation methods, we had shown that there are major changes in histone acetylation levels over these genes in cells corresponding to various developmental stages, but that the condensed chromatin domain and certain regulatory elements maintain their low and high acetylation levels (respectively) throughout development. We have now examined the distribution of histone H3 methylation over the same region. There is a striking correlation between methylation at lysine 4 and patterns of acetylation during development, and a remarkable anti-correlation of those two modifications with methylation of histone H3 at lysine 9. These results provide the first strong support in higher eukaryotes for a relationship between histone methylation over large regions and gene expression. We also found that the neighborhood of the beta- globin insulator element is characterized by histones H3 and H4 highly acetylated at all sites, H3 highly methylated at lysine 4, and nearly unmethylated at lysine 9. These observations have led us to a model for the way in which the insulator may protect against position effects, which is consistent with recent results on the propagation of condensed chromatin domains.